Nucleic acid encoding HB15 polypeptides

ABSTRACT

HB15-related lymphocyte activation antigens, and nucleic acid sequences encoding HB15-related antigens are disclosed. Also disclosed are antibodies reactive with HB15.

Part of the work leading to this invention was made with United StatesGovernment funds. Therefore, the U.S. Government has certain rights inthis invention.

This application is a continuation-in-part of U.S. patent applicationSer. No. 07/870,029 filed Apr. 17, 1992, now U.S. Pat. No. 5,316,920.

FIELD OF THE INVENTION

This invention relates to nucleic acid sequences encoding humanlymphocyte activation antigens, particularly to sequences encodinglymphocyte activation antigen HB15, and to the proteins and polypeptidesencoded by those sequences.

BACKGROUND OF THE INVENTION

The Ig gene superfamily, described by Williams et al., Annu. Rev.immunol., 88:381-405 (1988), encompasses a large family of genes thatare presumed to have evolved from a common precursor. Many of the Igsuperfamily members are involved in cell-cell adhesion and signaltransduction. In addition, many of the cell-surface molecules whichregulate immune responses contain conserved structural features similarto those found in immunoglobulin (Ig). While most members of the Ig genesuperfamily contain multiple linearly-assembled Ig-like domains, severalproteins have been identified that contain single Ig-like domains.

Single Ig-like domain proteins that are known or assumed to be involvedin cell-cell adhesion include: CD8α (Littman et al., Cell 40:237(1985)), CD8β (Johnson et al., Nature 323:74 (1986)), CD7 (Aruffo etal., EMBO J. 6:3313 (1987)), Thy-1 (Williams et al., Science 216:696(1982)), CD28 (Aruffo et al., Proc. Natl. Acad. Sci. USA 84:8573(1987)), CTLA-4 (Brunet et al., Nature 3.28:267 (1987)) and Po which isa structural protein of the peripheral myelin sheath (Lemke et al., Cell40:501 (1985)). In addition, other single Ig-domain containing proteinsassociate with the antigen receptors of B and T lymphocytes, formingmultimeric signal-transducing complexes. These include: CD3γ, δ and εchains (Gold et al., Nature 321:431-434 (1986); van den Elsen et al.,Nature 312:413-418 (1984)), CD79β (Hermanson et al., Proc. Natl. Acad.Sci., USA 85:6890 (1988)), and CD79β (Sakaguchi et al., EMBO J.7:3457-3464 (1988)).

Two proteins containing single Ig-like domains found on lymphocytes arepreferentially associated with cellular activation and are known to beinvolved in mediating cell-cell interactions. CD28 is expressed muchmore on activated than nonactivated T and B lymphocytes (Turka et al.,J. Immunol. 144:1646 (1990)), and CTLA-4 is expressed mostly, if notexclusively, by activated T and B lymphocytes (Brunet et al., Nature328:267 (1987); Harper et al., J. Immunol. 147:1037-1044 (1991)). Therole of CD28 as a T cell receptor for the CD80 molecule expressed byactivated B cells has been recently identified (Linsley et al., Proc.Natl. Acad. Sci. USA 87:5031-503 (1990); Freeman et al., J. Immunol.143:2714-2722 (1989)), as has a similar role for CTLA-4 (Linsley et al.,J. Exp. Med. 174:561-569 (1991)). As with CD28 and CD80, most of theIg-like domain-containing receptors interact with members of the Igsuperfamily present on other cells.

It is an object of the invention to provide a new member of the Ig genesuperfamily. Another object of the invention is to provide a proteinthat is found predominantly on lymphoid tissue. Yet another object ofthe invention is to provide a protein that contains an extracellularsingle Ig-like domain. Yet another object of the invention is to providea nucleic acid encoding the protein or a biologically active portion ofthe protein. Another object of the invention is to provide nucleic acidprobes for identifying the protein or homologs thereof. Yet anotherobject of the invention is to provide an antigen that is present onactivated lymphocytes, but absent on inactive lymphocytes and most othercell types.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a human lymphocyte cDNA whichencodes a novel glycoprotein present on activated lymphocytes, termedHB15 or CD83 (WHO nomenclature).

The invention thus features a nucleic acid isolate encoding thepolypeptide HB15 and able to hybridize to a nucleic acid encoding apolypeptide having an amino acid sequence shown in SEQ ID NO:2. HB15mammalian analog refers to a polypeptide which has a tissue distributionsimilar to human HB15, i.e., is present on activated lymphocytes anddendritic cells, and is encoded by a nucleic acid able to hybridize to anucleic acid encoding the amino acid sequence shown in SEQ ID NO:2."HB15 fragment" or "HB15 analog fragment" refers to a polypeptide of atleast 5 amino acids, preferably at least 10 amino acids, and mostpreferably at least 20 amino acids, which in its native context is partof a protein having the tissue distribution pattern of HB15. An HB15fragment or HB15 analog fragment will include a portion of HB15 such asone of the extracellular, transmembrane or cytoplasmic domains, or asmaller polypeptide, such as an immunogenic region of HB15.

In preferred embodiments, the nucleic acid isolate encodes a polypeptidethat is recognized by a monoclonal antibody specific for an HB15epitope. Preferably, the nucleic acid isolate encodes a polypeptidehaving the complete amino acid sequence shown in SEQ ID NO:2, or theportion of SEQ ID NO:2 comprising the HB15 extracellular domain (i.e.,amino acid numbers 1-125), the transmembrane domain (i.e., amino acidnumbers 126-147), or the cytoplasmic domain (i.e., amino acid numbers148-186). The boundaries of the mouse domains are approximately the sameas those of the humain domains, provided the sequences are aligned asshown in FIG. 6. Preferably, for polynucleotides greater than about 50bases, the nucleic acid isolate is hybridizable under stringentconditions to a portion of the nucleic acid sequence of SEQ ID NO: 1.For oligonucleotides less than about 50 nucleotides in length, thenucleic acid isolate is hybridizable under low stringency conditions,i.e., at about 42° C. in the presence of 30% formamide according toconditions described in Benton and Davis (1977, Science 196:180), herebyincorporated by reference. Preferably, the nucleic acid isolate isgreater than about 15 nucleotides, more preferably greater than about20, 50 or 100 nucleotides.

The invention also encompasses replicable expression vectors containingnucleic acid sequences encoding the HB15 protein or portions thereof,including an HB15 domain, as defined above, or immunogenic fragments,and host cells transfected with such a vector (e.g., for a bacterial,yeast, or eucaryotic cell culture).

The invention also encompasses HB15 or portions thereof which areimmunogenic, and thus useful as immunogens in order to raise antibodiesagainst HB15 or portions thereof including any of its specific domainsor fragments thereof.

The invention also features antibodies reactive with HB15 or fragmentsthereof.

The invention also features methods of producing human HB15 or amammalian homolog of human HB15, comprising transforming a host cellwith a nucleic acid encoding a polypeptide able to hybridize to asequence encoding the amino acid sequence shown in SEQ ID NO: 2,culturing the transformed cell, and recovering the HB15 protein orhomolog from the cell culture.

The invention also encompasses methods of detecting the presence ofhuman HB15 or of a mammalian HB15 analog on a cell, comprisingsubjecting a cell suspected of bearing HB15 on its surface to anantibody that recognizes HB15, and detecting binding of the antibody tothe cell.

The invention also features methods of producing a polypeptide encodedby a nucleic acid isolate greater than about 15 bp and capable ofhybridizing under low or high stringency conditions to a nucleic acidsequence shown in SEQ ID NO: 1. The method includes the steps of (a)providing cells which in the untransfected form do not express a nucleicacid isolate greater than about 15 bp and hybridizable to a nucleic acidsequence shown in SEQ ID NO: 1; (b) transfecting the cells with thenucleic acid isolate operably linked to suitable control sequences underconditions effective for the production of the encoded polypeptide; and(c) recovering the polypeptide.

The invention thus also features a polypeptide having HB15 biologicalactivity and encoded by a nucleic acid isolate able to hybridize underlow or high stringency conditions to a nucleic acid encoding apolypeptide having the amino acid sequence shown in SEQ ID NO: 2. Inaddition, the invention includes a polypeptide encoded by a nucleic acidisolate greater than about 15 nucleotides, hybridizable under low orhigh stringency conditions to the complement of the nucleic acidsequence shown in SEQ ID NO: 1.

The invention also features a purified nucleic acid molecule encoding anamino acid sequence of an HB15 molecule from an animal species otherthan human, the nucleic acid molecule being isolated by: (1) hybridizinga nucleic acid isolate with a population of nucleic acid molecules froman animal species other than human, preferably under low stringencyhybridization conditions, wherein the nucleic acid isolate encodes HB15or a portion thereof that is recognizable by a monoclonal antibodyspecific for an HB15 determinant, and is able to hybridize understringent conditions to a nucleic acid encoding a polypeptide having theamino acid sequence shown in SEQ ID NO: 2; (2) identifying a firstnucleic acid molecule to which the nucleic acid isolate stringentlyhybridizes; and (3) isolating the first nucleic acid molecule, whereinthe first nucleic acid molecule encodes a polypeptide having an aminoacid sequence shown in SEQ ID NO. 2.

This purified nucleic acid molecule may be further isolated by theadditional steps of: (4) hybridizing a nucleic acid isolate with apopulation of nucleic acid molecules from an animal species other thanhuman wherein said nucleic acid isolate encodes HB15 or is recognizableby a monoclonal antibody specific for an HB15 determinant, and is ableto hybridize to a nucleic acid encoding a polypeptide having the aminoacid sequence shown in SEQ ID NO: 2; (5) identifying a second nucleicacid molecule to which the nucleic acid isolate hybridizes; and (6)isolating the second nucleic acid, wherein the first and second nucleicacid molecules, joined together in an amino acid reading frame, encodean amino acid sequence of SEQ ID NO. 2.

Preferably, the nucleic acid molecule is a murine nucleic acid.

The invention also features an isolated nucleic acid able to hybridizeto the nucleic acid molecule described immediately above, andpolypeptides encoded by that nucleic acid molecule.

As used herein the term "identify" is intended to include techniquesthat require detection, isolation or purification of HB15 protein or itsencoding genetic material. The terms "isolated" and "essentiallypurified" refer to a nucleic acid or protein sequence that has beenseparated or isolated from the environment in which it was prepared orin which it naturally occurs.

Nucleic acid or protein sequences may be in the form of chimericmolecules, i.e., which lack one or more of the three domains found inthe native molecule, or chimeric hybrids in which one domain issubstituted with a domain from another type of molecule, e.g., a toxinor an Ig molecule. Examples of chimeric hybrids include but are notlimited to molecules which contain extracellular domains in which one ormore of these domains are heterologous. Such hybrids, e.g., animmunoglobulin fusion protein, are useful for promoting serum half-lifeor multimerization of the molecule to increase avidity. Truncated HB15molecules include but are not limited to HB15 comprising anextracellular domain free of transmembrane and cytoplasmic domains,which is useful for identifying a ligand or disrupting cell/cellinteraction, e.g., dendritic/T cell interactions.

The term "immunogenic fragment" refers to a fragment of HB15 that reactswith antibodies specific for a determinant of HB15.

The HB15 protein or immunogenic fragment can be used as antigenicreagents for immunization of a host animal in the preparation ofantibodies specific for HB15. An HB15 antibody may also be used todeliver drugs, toxins, or imaging agents to cells that express HB15.HB15 cDNA can be used to produce these proteins or peptide fragments; toidentify nucleic acid molecules encoding related proteins orpolypeptides (e.g., homologous polypeptides from related animal speciesand heterologous molecules from the same species); or to construct genesencoding other new, chimeric molecules. In addition, HB15 cDNA can beused to synthesize antisense oligonucleotides for inhibiting theexpression of the HB15 protein. Assays for HB15 production or expressionby cells are made possible by the development of monoclonal antibodiesselectively reactive with the HB15 protein.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the HB15 cDNA clone and the location ofrestriction sites, showing the extracellular domain ("extracell"), thetransmembrane domain ("TM"), and the 3' untranslated region (3'UT);

FIG. 2 shows the cDNA nucleotide sequence (SEQ ID NO: 1) and the deducedamino acid sequence (SEQ ID NO: 2) of HB15; the vertical arrowrepresents the predicted cleavage site for generation of the matureprotein; numbers shown above the amino acid sequence designate aminoacid residue positions of the putative mature protein; numbers to theright of the nucleotide sequence designate nucleotide positions; the *indicates the translation termination codon; underlined nucleotidesdelineate translated regions with hydrophobic character; underlinedamino acids indicate potential N-linked glycosylation attachments sites;wavy underlining delineates a poly (A) attachment signal sequence; aminoacids conserved in Ig-like domains are indicated by (+); cysteineresidues are circled; arrowheads below the nucleotide sequence denoteexon/intron boundaries;

FIG. 3 shows a hypothetical model for the structure of the extracellulardomain of HB15, cysteine residues are shown as filled in circles; aminoacids encoded by different exons are indicated by alternatively shadedcircles; numbers represent the predicted amino acid residue positions asshown in FIG. 2;

FIG. 4A shows immunofluorescence results obtained with threelymphoblastoid cell lines that express HB15 (A) with blood lymphocytesbefore and after mitogen activation (B); open histograms show cellularreactivity with the HB15a antibody; shaded histograms representbackground levels of immunofluorescence staining obtained withunreactive control antibodies;

FIG. 4B shows immunofluorescence results obtained with blood lymphocytesbefore and after mitogen activation (B), with open and shaded histogramsrepresented as in FIG. 4A;

FIG. 5A shows immunohistochemical analysis of HB15 expression in tonsiland lymph node cells;

FIG. 5B shows immunohistochemical analysis of HB15 expression ingerminal centers;

FIG. 5C shows immunohistochemical analysis of HB15 expression ininterfollicular regions (i.e., the T-cell zone);

FIG. 5D shows immunohistochemical analysis of CD1 expression in asubpopulation of dendritic cells;

FIG. 5E shows immunohistochemical analysis of HB15 expression in asubpopulation of thymic medulla cells; and

FIG. 5F shows immunohistochemical analysis of HB15 expression in asubpopulation of dendritic cells (skin Langerhan's cells).

FIG. 6 is a comparison of human and mouse cDNA sequences encoding HB15.

FIG. 7 presents sequence locations of oligonucleotide probes used forPCR amplification of human and mouse HB15 cDNAs relative to the humanand mouse HB15 DNA sequences.

FIG. 8A shows results of PCR amplification and gel electrophoresis ofamplified fragments.

FIG. 8B shows results of Southern blots of the gels shown in FIG. 8Ausing a probe from the HB15 transmembrane domain.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The lymphocyte activation antigen, HB15, is expressed virtuallyexclusively by lymphoid tissue and skin Langerhans cells. HB15 is asingle-chain cell-surface glycoprotein of M_(r) 45,000. Referring toFIG. 1, the structural features of the HB15 protein, predicted fromnucleotide sequence derived from multiple cDNA clones, establish it as anew member of the Ig superfamily. The predicted structure of HB15 isthat of a typical membrane glycoprotein with a single extracellularIg-like domain, a transmembrane domain and an approximately 40 aminoacid cytoplasmic domain.

cDNA cloned from a human lymphocyte library were analyzed and shown toencode the novel cell-surface glycoprotein HB15, expressed by activatedlymphocytes. The mature 186 amino acid protein encoded by the cDNA wascomposed of a single extracellular V type immunoglobulin (Ig)-likedomain, a transmembrane domain and a 39 amino acid cytoplasmic domain.Northern blot analysis revealed that HB15 derives from three mRNAtranscripts of ˜1.7, 2.0 and 2.5 kb expressed by lymphoblastoid celllines. It is likely that the entire coding region for HB15 wasidentified, as transfection of cell lines with the pHB15 cDNA generatedcell surface expression of the protein and the M_(r) of theimmunoprecipitated protein was similar in both cDNA transfected cells(˜45,000) and HB15⁺ Raji cells (˜40,000). It is also likely that HB15undergoes extensive post-translational processing, as HB15 was expressedas a single chain molecule, yet the determined M_(r) was twice thepredicted size of the core protein. Since HB15 was also expressed on thesurface of cDNA transfected cells, including COS cells, CHO cells, amouse pre-B cell line and a human erythroleukemia line, it is likelythat surface expression is not dependent on expression of othercomponents of a molecular complex as occurs with the Ig-like proteinsthat associate with the T and B cell antigen receptors.

Monoclonal antibodies reactive with HB15 were produced and used to showthat HB15 expression is specific for lymphoblastoid cell lines andmitogen-activated lymphocytes; HB15 was not expressed at detectablelevels by circulating leukocytes. Immunohistological analysis revealedthat HB15 has a unique pattern of expression among tissues, being foundpredominantly in hematopoietic tissues with scattered expression byinterfollicular cells and weak expression by mantle zone and germinalcenter cells. Uniquely, HB15 is also expressed by Langerhans cellswithin the skin and circulating dendritic cells. Thus, the HB15glycoprotein represents a new member of the Ig superfamily.

Comparison of the HB15 amino acid sequences (SEQ ID NO: 2) with otherpreviously identified proteins did not reveal any striking homologies,except the similarity of the extracellular Ig-like domain with othermembers of the Ig superfamily. The HB15 Ig-like domain contained many ofthe conserved features found in the V-set of domains, as shown in FIG. 2(Williams et al., Ann. Rev. Immunol. 88:381-405 (1988)). Based on thehomology with Ig domains, HB15 is likely to possess a disulfide bondlinking Cys 16 and Cys 88. This would place 71 amino acids between thetwo Cys residues which is of the appropriate size for V-related domains(Williams et al., supra). There is the potential for additionaldisulfide bond formation between residues at positions 8, 81 and 110since these Cys are present in the extracellular domain as well. Inaddition, HB15 has a Cys residue located within the predicted membranespanning domain at position 144. Cys residues are also located atidentical positions in CD3δ and CD7, suggesting some functionalsignificance, perhaps as sites for fatty acylation (Kaufman et al., J.Biol. Chem. 2.59:7230-7238, (1984); Rose et al., Proc. Natl. Acad. Sci.,USA 81:2050-2054 (1984)). The HB15 cytoplasmic tail is similar in sizeto that of CD7 (Aruffo et al., EMBO J. 6:3313 (1987)), but shared noamino acid sequence similarity with known proteins. However, the fiveSer/Thr residues within this domain could serve as potential sites ofphosphorylation. Thus, HB15 appears to be a newly described lymphocytecell surface antigen that shares no apparent relatedness with previouslydescribed structures.

The HB15 extracellular domain is different from the typical Ig-likedomain in that it is encoded by at least two exons. Analysis of partialgenomic DNA sequence revealed that half of the Ig-like domain is encodedby a single exon and the putative membrane spanning domain is alsoencoded by a distinct exon (FIG. 2). That Ig-like domains can be encodedby more than one exon has been observed for some members of the Igsuperfamily, including the Po protein (Lemke et al., Neuron 1:73-83(1988)), CD4 (Littman et al., Nature 325:453-455 (1987)) and N-CAM(Owens et al., Proc. Natl. Acad. Sci., USA 84:294-298 (1987)). Thisfinding supports structural analyses which suggested that Ig domains mayhave arisen from an ancestral half-domain that evolved throughduplication and subsequent adjoining. However, each of the above genesand the HB15 gene contain introns at different locations between thesequences coding for the conserved Cys residues of the disulfide bond(Williams et al., Annu. Rev. Immunol. 88:381-405 (1988)). This findingsupports the notion that introns may have been subsequently inserted tointerrupt the ancestral Ig-like domain at later points during theevolution of each of these domains.

Expression of HB15 appears to be generally restricted to lymphocytessince two monoclonal antibodies reactive with HB15 failed to detect HB15on most other hematopoietic cells. HB15 expression may be a late eventin lymphocyte development as most thymocytes and circulating lymphocytesdid not express detectable levels of cell surface HB15. However, afterbeing activated by mitogens, peripheral lymphocytes expressed maximallevels of cell surface HB15 on days 3 through 5, the period of timeduring which maximal proliferation occurred. HB15 may be expressed atlow levels by monocytes, especially after culture or activation, but thelevel of expression is low and may just result from Fc receptor mediatedantibody attachment. Many T and B cell lines also expressed HB15, butexpression was generally at low levels. Interestingly, cell-surface HB15expression by cell lines was highest during periods of maximalproliferation such as on the first day after the cultures were fed.These results imply that HB15 is important for maximal growth oflymphoblastoid cells or the maximal growth of cells is critical for theexpression of this antigen. This was consistent with the observationthat HB15 was expressed by germinal center cells in hematopoietictissues. Nevertheless, HB15 expression appeared to be lymphoid tissuerestricted as revealed by immunohistological analysis of twenty-twodifferent tissues. The only exception was the finding that skinLangerhans cells express HB15. This unique pattern of restrictedexpression, along with the structural analysis of the protein, indicatesthat HB15 is a newly identified lymphocyte activation antigen.

The structural similarity of HB15 with other members of the Igsuperfamily suggests that it may be involved in cellular interactions,since Ig-like domains are frequently involved in a variety of homotypicand heterotypic interactions in the immune and nervous systems. Theseinteractions include binding functions that trigger a subsequent eventbelow the cell surface or adhesion. A key functional feature is thathomophilic or heterophilic binding usually occurs between Ig-relatedmolecules, and this is often between molecules on opposed membranesurfaces. The structural relatedness of HB15 to these other proteins mayimply a role for this lymphocyte activation protein in either homotypicor heterotypic interactions of lymphocytes following activation of otherHB15⁺ cell types. As used herein, "homophilic" refers to cells of thesame type that have a specific association or attraction for each other;"homotypic" refers to two molecules or cells of the same form thatinteract in a specific fashion; "heterophilic" refers to cells ofdifferent types having a specific association with each other; and"heterotypic" refers to two molecules or cells of different types thatinteract in a specific fashion.

It is understood that the particular nucleotide (SEQ ID NO: 1) and aminoacid (SEQ ID NO: 2) sequences disclosed in FIG. 2 are representative ofthe human counterpart, and that related mammalian genes and theirencoded proteins can be obtained following the teachings of thisdisclosure, as demonstrated herein for isolation of the mouse HB15homolog. A mammalian homolog of the sequences disclosed in FIG. 2 willinclude a gene which is identified under stringent hybridizationsconditions using a probe based on an approximately 20 nucleotide regionof sequence identity between the FIG. 2 nucleotide sequence and the geneencoding the mammalian homolog. For example, cross-hybridization of thedisclosed nucleic acid sequences with genetic material from human cells,can readily be performed to obtain equivalent human sequences; forexample, see the oligonucleotide sequences presented in Table 1. In ananalogous manner, degenerate oligonucleotides can readily be synthesizedfrom the disclosed amino acid sequence, or portions thereof, andamplified using any well-known amplification technique, such as thepolymerase chain reaction, to obtain probes that bind to equivalenthuman sequences. Proteins or polypeptides encoded by equivalentsequences can be produced. Antibodies directed against the disclosedprotein or peptides can also be raised and employed to cross-react withhuman and other mammalian peptides having similar epitope(s). Thosepeptides isolated in this manner that have similar antibody reactivitypatterns to those of the disclosed proteins or peptides are consideredequivalents of the disclosed proteins or peptides.

The following examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These examples are not intended in any way otherwise tolimit the scope of the disclosure.

EXAMPLE I

Human cDNA clones encoding HB15 were isolated and the encoded human HB15protein characterized, as follows.

A human tonsil cDNA library was screened by differential hybridization(see Tedder et al., Proc. Natl. Acad. Sci., USA 85:208, 1988) , herebyincorporated by reference using labeled cDNA from the B lymphoblastoidcell line Raji and the T cell line H-SB2. Two of the 261 RAJI⁺ H-SB2⁻cDNA clones isolated, pB10 (˜2.5 kb) and pB123 (˜1.2 kb), crosshybridized, yet failed to hybridize with cDNA that encode known B cellsurface antigens (Tedder et al., supra).

Expression of the mRNA was examined by Northern blot analysis usingpoly(A)⁺ RNA isolated from B cell lines (NALM-6, Namalwa, Daudi, SB, andRaji), T cell lines (Hut-78, H-SB2, and MOLT-3) and the erythroleukemialine, K562. Poly(A)⁺ RNA was isolated as described (Maniatis et al.,Molecular Cloning: A Laboratory Manual, (1982)). For Northern-blotanalysis, 2 μg of poly(A)⁺ RNA was denatured with glyoxal, fractionatedby electrophoresis through a 1.1% agarose gel and transferred tonitrocellulose (Thomas, Methods Enzymol. 100:255 (1983)). The pB123 cDNAinsert used as probe was isolated, nick-translated (Rigby et al., J.Mol. Biol. 113:237-251 (1977)) and hybridized with the filters asdescribed (Wahl et al., Proc. Natl. Acad. Sci., USA 76:3683-3687(1979)). Hybridization at high stringency was with 50% (v/v) formamide,4× SSC, 10% (w/v) Na dextran sulfate at 42° C. The filters were washedat 65° C. with 0.2× SSC, 0.1% SDS. RNA size was determined by comparisonwith 28S and 18S ribosomal RNA run on the same gels as standards. Thesame blot was also hybridized with cDNA clones containing a housekeepingmRNA of unknown identity revealing that all mRNA were intact and weresimilar in quantity of this expressed mRNA. For hybridization at lowstringency the conditions are overnight incubation at 42° C. in asolution comprising: 20% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardts solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.

The pB123 cDNA hybridized strongly with three mRNA species of ˜1.7, ˜2.0and ˜2.5 kb in SB and Raji. Daudi and Namalwa cells expressed lowerlevels of this mRNA. Further autoradiography of the blot (7 days)revealed that the NALM-6, Hut-78 and MOLT-3 cells also expressed thesethree mRNA species, but at much lower levels, and faint hybridizationwith H-SB2 RNA was detected. These results suggested differentialexpression of this gene among leukocyte subpopulations.

Restriction maps were generated for these cDNA, as described by Maniatiset al., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring HarborPress, CSH, N.Y., and their nucleotide sequences determined as describedSanger et al., Proc. Nat. Aca. Sci. 74:5463, 1977. Both cDNA wereoverlapping and contained open reading frames at their 5' ends with thepB123 cDNA having the longest 5' sequence. Since neither clone containeda translation initiation site, the pB10 cDNA insert was used to isolate13 additional cross-hybridizing cDNA from a human tonsil library. ThepB10 insert was purified, labeled by nick translation (Rigby et al., J.Mol. Biol. 113:237-251 (1977)) and used to isolate homologous cDNA byagain screening the same human tonsil cDNA library in λgt11 (Weis etal., Proc. Natl. Acad. Sci., USA 83:5639-5643 (1986)) as described (Zhouet al., Immunogenetics 35:102-111 (1992)). Positive plaques wereisolated, cloned and the cDNA inserts were removed by EcoR I digestionand subcloned into pSP65 (Melton et al., Nucleic Acids Res. 12:7035-7056(1984)). Restriction maps and nucleotide sequence determinationindicated that 12 of the cDNA were overlapping, with one cDNA having thelongest sequence at the 5' end. The restriction map and nucleotidesequence of this clone, termed pHB15, is shown in FIG. 1. The fulllength cDNA clone is likely to include an ˜500 bp fragment at the 3' endthat was removed from the cDNA by EcoR I digestion and subcloning. Eightother independent cDNA clones had similar EcoR I generated fragments andan EcoR I site was located at the identical nucleotide position in allcDNA that were sequenced.

The pHB15 cDNA had a 625 bp open reading frame, with the major portionof the cDNA representing untranslated sequence. The determinednucleotide sequence (SEQ ID NO: 1) and predicted amino acid sequence(SEQ ID NO: 2) of HB15 are given in FIG. 2. The predicted cleavage siteused to generate the mature protein is shown by a vertical arrow. Thenumbers shown above the amino acid sequence designate amino acid residuepositions of the putative mature protein and the numbers on the rightdesignate nucleotide positions. Amino acids are designated by thesingle-letter code, and * indicates the termination codon. Nucleotidesdelineating translated regions with hydrophobic character areunderlined. Amino acids indicating potential N-linked glycosylationattachment sites are underlined. A poly(A) attachment signal sequence isindicated by wavy underlining. The Cys residues are circled and aminoacids which are often conserved in Ig-like domains are indicated by (+).Arrow heads below the nucleotide sequence denote exon/intron boundariesidentified in another DNA clone.

The first ATG shown is the most likely initiation codon for translationsince it conforms to the proposed translation initiation consensussequence, (A/G)CCAUG (Kozak, Cell 44:283-292 (1986)). It is likely thatthe different mRNA species result from differential use of poly(A)attachment sites, AATAAA, since one was found at nucleotide position1248 in the middle of the 3' untranslated region (FIG. 2). This poly(A)attachment site was functional in the pB123 cDNA since it was followedby a poly(A) tail. A poly(A) attachment site or tail was not found inthe ˜550 bp EcoR I fragment which presumably represents the 3' end ofthe pHB15 cDNA.

One clone isolated from the cDNA library (˜3.0 kb long) that hybridizedwith the pB123 cDNA had a unique sequence with 229 and 107 bp longsegments that were identical to those found in the other cDNA. Theseregions had flanking sequences that corresponded to the consensus 5' and3' splice sequences which demark exon boundaries (Aebi et al., TrendsGenet. 3:102-107 (1987)) indicating that this aberrant cDNA was composedof introns and two exons. The three splice junction sites identified bythis clone are shown (FIG. 2).

The predicted length of the HB15 protein was 205 amino acids (FIG. 2).However, the pB123 cDNA was missing the codon AAG at nucleotide position500 so the protein may be one amino acid shorter in some cases. This mayresult from differential splicing at an exon/intron border, that resultsin the inclusion or loss of a codon since this codon abuts a potentialsplice site. A similar phenomenon has been found in the CD19 gene whichalso encodes a member of the Ig superfamily (Zhou et al., Immunogenetics35:102-111 (1992)). Hydropathy analysis of the HB15 amino acid sequenceby the method of Kyte et al., J. Mol. Biol. 157:105 (1982) revealed tworegions of strong hydrophobicity. The first hydrophobic stretch of 19amino acids represents a typical signal peptide at the amino terminalend of the protein. The algorithm of von Heijne, Nucleic Acids Res.14:4683-4690 (1986) predicts that the most probable amino-terminus ofthe mature protein would be the Thr following amino acid 19. The secondhydrophobic region of 22 amino acids most probably represents thetransmembrane region. Three potential N-linked glycosylation attachmentsites (N-X-S/T) were found in the extracellular domain. Therefore, thepredicted molecular mass of the core protein would be ˜20,500.

Six Cys residues were found in the extracellular domain of HB15 and onein the putative membrane spanning domain. One pair of these residues atpositions 16 and 88 delineate Ig-like domains (Williams et al., Annu.Rev. Immunol. 88:381-405 (1988)). This domain contained many of thehallmark amino acids which define the V set of Ig-like domains. Acomputer search of nucleotide and protein sequences was conducted usingthe Protein Identification Resource Data (GenBank release 66 andSwiss-Prot-16). Gap penalties of -1 were assessed during sequencehomology analysis for each nucleotide or amino acid in the sequencewhere a gap or deletion occurred. The computer search of proteinsequences showed that no proteins shared significant sequence homologywith HB15 other than some members of the Ig superfamily.

Referring to FIG. 3, a hypothetical model is given for the structure ofthe extracellular domain of HB15 based on the proposed arrangement ofthe β-pleated sheets for the V domain of Ig heavy chain. Cys residuesare represented as filled circles and amino acids encoded by differentexons are indicated by alternatively shaded circles. Numbers representthe predicted amino acid residue positions as in FIG. 2.

EXAMPLE II

Preparation of HB15 Truncated and Chimeric Molecules.

Variant forms of HB15, e.g., truncated molecules or chimeric (i.e.,hybrid) molecules containing substituted domains, may be prepared usingconventional recombinant DNA techniques known to those of skill in theart and the HB15 nucleotide sequences (SEQ ID NO: 1--human and SEQ IDNO: 3--mouse) and amino acid sequences (SEQ ID NO: 2--human and SEQ IDNO: 4--mouse ) disclosed herein. See Maniatis et al., 1982, MolecularCloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., herebyincorporated by reference.

A chimeric HB15 molecule is one in which one or two of theextracellular, transmembrane, and cytoplasmic domains is removed andreplaced by the corresponding domain from another species, e.g., adomain from the mouse sequences disclosed herein.

A truncated HB15 molecule is one in which a portion of the molecule hasbeen deleted. Truncated molecules will include those molecules in whichone or both of the transmembrane and cytoplasmic domains has beendeleted from the molecule, leaving, minimally, the extracellular domainor a portion thereof. A truncated HB15 molecule may be used to constructa protein in which the truncated HB15 end is fused to an effectormolecule such as a drug toxin, or imaging agent using conventionalmethods for joining such molecules at the DNA or polypeptide level.

For example, a truncated form of HB15 may include the extracellulardomain free of the cytoplasmic and transmembrane domains. Thisrepresentative truncated HB15 molecule may be constructed by cleaving aDNA fragment containing a nucleotide sequence encoding the extracellulardomain using standard PCR amplification to amplify that region. Theamplified fragment then may be ligated to compatible ends of anexpression vector and transfected into a host cell, e.g., an activatedlymphocyte, which allows for production of the encoded domain. Truncatedmolecules containing other portions of the HB15 molecule may beconstructed using conventional PCR amplification procedures. One or moreof these sites may be utilized, depending upon which domains of the HB15molecule are preferred.

Chimeric forms of HB15 also may be constructed using conventionalrecombinant DNA technology and the nucleotide sequence (SEQ ID NO:1--human and SEQ ID NO: 3--mouse) and amino acid sequences disclosedherein. For example, where a chimeric molecule comprising humanextracellular and transmembrane HB15 domains and a murine cytoplasmicdomain is desired, the human domains may be isolated using restrictionenzymes which generate those portions of human HB15 and joined to amurine cytoplasmic domain using cloning techniques, and expressed asdescribed above for truncated molecules.

EXAMPLE III

Isolation of Mammalian Homolog of HB15.

A nucleotide sequence encoding HB15 from another mammalian species maybe isolated by first hybridizing a nucleic acid probe with a populationof nucleic acid molecules from an animal species other than human underhybridization conditions sufficient to allow for annealing of the probeto a homologous region of the target gene. The nucleic acid probe mayencode full-length human HB15 or a fragment thereof; the encodedpolypeptide will be recognizable by a monoclonal antibody specific foran HB15 determinant, and will be able to hybridize to a nucleic acidencoding a polypeptide having the amino acid sequence shown in SEQ IDNO: 2. The probe will thus identify a first nucleic acid molecule towhich the probe preferably stringently hybridizes. The first nucleicacid molecule then may be isolated and will thus encode a polypeptidehaving an amino acid sequence shown in SEQ ID NO. 2.

If a partial HB15 molecule, e.g., a heterologous domain is isolated inlieu of an entire HB15 molecule, a second nucleic acid molecule to whichthe nucleic acid probe preferably stringently hybridizes may beidentified and isolated, wherein the first and second nucleic acidmolecules, joined together in an amino acid reading frame, encode anamino acid sequence of SEQ ID NO. 2.

Alternative strategies may also be used for isolating a mammalian HB15homolog. For example, the mouse HB15 homolog was isolated as follows.

The mouse HB15 gene was isolated by screening a murine genomic libraryby cross-hybridization with a 1.7 kb subclone of the human HB15 cDNAunder low stringency conditions.

Genomic DNA clones were isolated from a genomic DNA library made withpartial Mbo I-digested mouse genomic DNA that was isolated from a 129Svmouse strain and inserted into the vector lambda-DASH II (Stratagene, LaJolla, Calif.). The human HB15 cDNA clone was labeled by nicktranslation and used to screen the mouse genomic DNA library accordingto the method of Benton and Davis (1977, Science 196f180). Hybridizationwas performed at 42° C. in the presence of 30% formamide and the filterswere finally washed at 50° C. in 1× SSC with 0.1% SDS (w/v). The humanHB15 cDNA probe contained the entire protein coding sequence and theentire 3' untranslated regions. Positive plaques were isolated, andphage DNA were characterized by restriction enzyme mapping as described(Maniatis et al., 1982, Molecular Cloning, supra). DNA fragments ofthese clones were generated by EcoR I or Hind III digestions and weresubcloned into the plasmids pSP65 or pSP64. Detailed restriction enzymemaps of the subclones were made. Exons were located by Southernhybridization analysis of endonuclease digested mouse genomic DNA clonesusing labeled human cDNA or oligonucleotide probes. Nucleotide sequenceswere determined by the dideoxy chain termination method (Sanger et al.,1977, Proc. Nat. Aca. Sci. 74:5463).

Overlapping mouse genomic clones spanning 23 kb contained most of themouse HB15 gene, from the 3' half of the immunoglobulin domain throughthe 3' untranslated region. Sequence analysis of the 3' portion of theimmunoglobulin-like domain, the transmembrane region, and thecytoplasmic domain demonstrated a significant degree of conservationbetween human and mouse sequences, such that amino acid identity is ˜70%in these exons (FIG. 6). Likewise, the 3' untranslated region contained1600 bp of extremely well conserved nucleotide sequence.

FIG. 6 shows the nucleotide sequence of mouse HB15 (m) (SEQ ID NO: 3)compared with the human (h) cDNA sequence. The precise nucleotidesequence for the 5' region of the mouse HB15 protein is not definitiveas indicated by nucleotides in lower case print. "*" indicates identityin nucleotide sequences between human and mouse. "-" indicates gaps inthe nucleotide sequence introduced to generate the highest levels ofhomology. The predicted cleavage site used to generate the matureprotein is shown by a vertical arrow. The numbers shown above the aminoacid sequence designate amino acid residue positions of the mature humanprotein and the numbers on the right designate nucleotide positions forthe human cDNA. Nucleotides delineating translated regions withhydrophobic character (leader and transmembrane domains) are doubleunderlined. Amino acids indicating potential N-linked glycosylationattachment sites are underlined. A poly(A) attachment signal sequence isindicated by wavy underlining. Amino acids which are often conserved inlg-like domains are indicated by (+). Arrow heads below the nucleotidesequence denote exon/intron boundaries identified in genomic DNA clones.

The 5' portion of mouse HB15 was isolated by PCR amplification of amouse B lymphocyte cDNA library using a 5' oligonucleotide sense probehomologous with the flanking sequence of the vector and using a 3'antisense oligonucleotide probe (#2489 in Table 1) homologous to the 5'half of the Ig like domain of mouse HB15. This generated anapproximately 400 bp cDNA fragment that was subcloned and sequenced. Thenucleotide sequence of the PCR product revealed that it was nearlyidentical in sequence to the human HB15 cDNA (FIG. 6). RNA was isolatedby a modification of the single step acid-guanidine-phenol-chloroformmethod from the mouse B cell line A20. One microgram of this RNA wasused to synthesize cDNA using random hexamer primer oligonucleotides andSuperscript reverse transcriptase (Bethesda Research Laboratories). ThecDNA synthesis reaction mixture contained 10mM Tris-HCl (pH 8.3), 50 mMKCl, 1.5 mM MgCl₂, and 0.8 mM each of dATP, dGTP, dCTP, and dTTP (Sigma,St. Louis, Mo.). 500 ng of the hexamer primer, 200 U of reversetranscriptase, and 1 μ of RNasin (Promega) were added to give a finalvolume of 25 μ. After 1 hour at 37° C. this reaction mixture was stoppedby heating to 95° C. for 5 min and then cooled to 4° C. for 5 min. 5 μof this reaction mixture was used to perform polymerase chain reactions(PCR) by adding 10 μ of PCR buffer, 50 pmol sense and antisense primersand amplification was carried out for 30 cycles as follows: denature for1 min. at 94° C., anneal for 1 min. at 55° C. and extend for 1 min. at72° C.

The PCR amplified cDNAs were electrophoresed through 1% agarose gels andtransferred to nitrocellulose. DNA size was determined byco-electropheresis of a 1-kb ladder (Bethesda Research Laboratories,Gaithersberg, Md.). Hybridization was performed at 50° C. in buffercontaining a 5' end-labeled oligonucleotide, 6×NET (3M NaCl, 0.02 mMEDTA, 0.15 mM Tris-HCl ph 8.3), 10× Denhardt's solution, 0.1% SDS (w/v),20 mM sodium phosphate, and 100 μg/ml salmon sperm DNA (Sigma). Filterswere finally washed in 2× SSC at room temperature. Autoradiography wasat room temperature for 30 min.

Within the immunoglobulin-like domain of human and mouse HB15, allcysteine residues have been conserved, including those which delineatethe immunoglobulin-like domain in the human protein. Partialdetermination of intron/exon boundaries for the mouse HB15 gene hasconfirmed that, as with the human HB15 gene, the immunoglobulin-likedomain in the mouse is encoded by at least two exons.

Mouse HB15 sequence-specific oligonucleotide primers generated from aportion of the immunoglobulin-like domain (#2406 in Table 1) and fromthe cytoplasmic domain (#2407 in Table 1) have been used as probes toexamine the pattern of expression of HB15 in mouse. The presence ofHB15-specific mRNA in spleen, kidney, liver, brain, muscle, lung,thymus, and thyroid tissue was tested by reverse transcriptase PCR andgenerated the expected DNA products in all organs. The identification ofHB15 mRNA in multiple organ sites may reflect the presence of dendriticcell family members present as a network of supportive or accessorycells in diverse tissue types throughout the body.

HB15 cDNAs were isolated from mRNA as follows. cDNA was produced fromRaji mRNA to determine whether oligonucleotides representing differentdomains of the molecule (FIG. 7) could be used as probes to generateHB15 nucleotide sequences. In FIG. 7, locations of oligonucleotides usedfor PCR amplification of cDNA are given. Oligonucleotides identical tothe human sequence are shown above the human cDNA while oligonucleotidesidentical to the mouse sequence are below the human cDNA sequence. The5' end of the oligonucleotide is indicated by an arrowhead; > for senseprimers and < for antisense primers. cDNA was amplified by PCR and theresulting products were characterized by Southern blot analysis withprobes that would hybridize with internal HB15 sequence. Both the entireopen reading frame and the 5' and 3' ends of cDNAs were amplified usingthe strategy shown in FIGS. 8A and 8B. In FIG. 8A, HB15 cDNAs weregenerated from RNA isolated from the Raji B cell line and the cDNAs wereamplified using appropriate combinations of a sense oligonucleotide andantisense oligonucleotide, whose sequences are defined in Table 1 asfollows: 1. #2083 and 2407; 2. 2406 and 2407; 3. 2085 and 2407; 4.LJZ001 and 2086; 5. LJZ001 and 2489; 6. LJZ001 and 2084; 7. LJZ001 andLJ33; 8. LJZ001 and TFT617; 9. LJZ001 and 2407. This strategy generatedcDNA fragments representing the 5' end or 3' end of the HB15 codingregion. FIG. 8A shows representative results from one experiment showingthe PCR amplified cDNAs obtained; PCR-generated cDNAs wereelectrophoresed on an agarose gel with DNA size markers and stained withethidium bromide. In FIG. 8B, Southern blots of replicates of the gel inA were probed with the end-labeled #2082 oligonucleotide.Autoradiographic results are shown. There were additional bands variablyobserved in some PCR reactions, but these bands were also seen incontrol reactions carried out with mRNA from HB15 negative cell lines(data not shown). These bands also failed to hybridize with internal32P-labeled oligonucleotide probe in most cases. Therefore, it is mostlikely that these minor species of PCR products represented artifact DNAgenerated in the PCR amplification process and do not represent realmRNA species.

                                      TABLE 1                                     __________________________________________________________________________    Probe                                                                             Human/Mouse                                                                          Orientation                                                                         Domain                                                                             Sequence                                                __________________________________________________________________________    LJZ001                                                                            m      sense Leader                                                                             GCC ATG TCG CAA GGC CTC CAG CTC C                       2086                                                                              h      antisense                                                                           5'1g exon                                                                          AC ACG GTC TCC TGG GTC AAG                              2084                                                                              h      antisense                                                                           3'1g exon                                                                          AC CTA AGT GGC AAG GTG ATC                              2085                                                                              h      sense 3'1g exon                                                                          GA CAG CAC TAT CAT CAG AAG                              2406                                                                              m      sense 3'1g exon                                                                          C TGC AGC TCG GGC ACC TAC AGG TG                        2489                                                                              m      antisense                                                                           3'1g exon                                                                          C TGC AGC TCG GGC ACC TAC AGG TG                        2083                                                                              h      sense TM exon                                                                            T GCA CAG CGT AAA GA                                    LJ33                                                                              h      antisense                                                                           TM exon                                                                            ACT TTT AAG AAA TAC AGA GCG GAG ATT GTC CT              TFT617                                                                            h      antisense                                                                           TM exon                                                                            G AAA TAC AGA GCG GAG ATT GTC CT                        2082                                                                              h      antisense                                                                           TM exon                                                                            ACA CTC ATC ATT TTC ACT TGT                             2407                                                                              m      antisense                                                                           cyto.tall                                                                          A GCT TTT CTT CCA GTC ACC TCC CCA                       __________________________________________________________________________                          A                                                   

EXAMPLE IV

Production of monoclonal antibodies reactive with HB15.

A monoclonal antibody reactive with HB15 or an HB15 homolog or portionthereof, particularly a portion of the extracellular domain of themolecule, may be prepared as described below for preparation of theanti-HB15a and anti-HB15b antibodies.

1. Preparation of Anti-HB15a and Anti-HB15b Antibodies

Hybridomas were generated by the fusion of NS-1 myeloma cells withspleen cells obtained from mice immunized with pHB15 cDNA-transfectedCOS cells. COS cells were transfected with the pHB15 cDNA insertsubcloned into a modified CDM8 vector (Aruffo et al., EMBO J. 6:3313(1987); Tedder et al., J. Immunol. 143:712-717 (1989)) using theDEAE-dextran method as described (Aruffo et al., EMBO J. 6:3313 (1987)).Cell surface expression was examined after 48 hours by indirectimmunofluorescence. Stable cDNA transfected cells were produced usingthe pHB15 cDNA cloned into the BamH I site of the retroviral vectorpZipNeoSV(X) in the correct orientation (Cepko et al., Cell 37:1053-1062(1984)). The murine pre-B cell line, 300.19, and the humanerythroleukemia cell line, K562, were transfected with this vector byelectroporation with subsequent selection of stable transfectants usingG418 (Gibco/BRL). Cells expressing HB15 were further enriched byreacting the cells with monoclonal antibodies with the subsequentisolation of HB15⁺ cells by panning on anti-mouse Ig coated plates. Celllines were grown in RPMI 1640 medium containing 10% fetal calf serum andantibiotics. Cultures of all cell lines were split the day beforeanalysis and were in logarithmic growth.

Anti-HB15 mAb were generated as described (Tedder et al., J. Immunol.144:532-540 (1990)) by the fusion of NS-1 myeloma cells with spleencells from BALB/c mice that were repeatedly immunized with COS cellstransfected with the HB15 cDNA. Each hybridoma was cloned twice and usedto generate ascites fluid. The isotypes of the mAb were determined usinga mouse monoclonal antibody isotyping kit from Amersham (ArlingtonHeights, Ill.).

Monoclonal antibodies reactive in indirect immunofluorescence assayswith HB15 mRNA positive cell lines, but not with HB15 negative celllines, were isolated. Two of these antibodies, anti-HB15a (IgG_(2b)) andanti-HB15b (IgG₃) also reacted with COS cells transfected with the pHB15cDNA, but did not react with cells transfected with CD19 cDNA (Tedder etal., J. Immunol. 143:712-717 (1989)) or the expression vector alone. Inaddition, these antibodies reacted with a human erythroleukemia cellline, K562, and a mouse pre-B cell line, 300.19, stably transfected withthe pHB15 cDNA. The antibodies did not react with untransfected parentcells, cells transfected with vector alone; or CD19, CD20 (Tedder etal., Proc. Natl. Acad. Sci., USA 85:208 (1988)) or LAM-1 (Tedder et al.,J. Exp. Med. 170:123-133 (1989)) cDNA transfected cells. In all cases,the reactivities of the anti-HB15a and anti-HB15b mAb were identical.

2. Mapping of HB15 Epitopes

A monoclonal antibody specific for a given region of HB15 may be madeusing a peptide corresponding to the region of the molecule as animmunogen, and using conventional hybridoma production procedures. Inaddition, the cross-reactivity of such antibodies can be ascertained asfollows. For example, the HB15a and HB15b mAb identify differentepitopes on the HB15 molecule. The HB15a mAb was conjugated to FITC(HB15a-FITC). K562 cells transfected with the HB15 cDNA were firstreacted with saturating amounts of either the HB15a or the HB15b mAb inthe form of diluted ascites fluid. After the appropriate incubationperiod, the cells were subsequently washed and then treated withHB15a-FITC. After the appropriate incubation period, the cells werewashed again to remove unbound HB15a-FITC and analyzed byfluorescence-based flow cytometry. Cells pretreated with HB15a mAb didnot bind HB15a-FITC since the unlabeled mAb blocked the binding of thelabeled reagent. In contrast, treatment of the cells with HB15b mAb hadno effect on the staining of the test cells with the HB15a-FITC. Theseresults demonstrate that the HB15a mAb binds to a different epitope ofthe HB15 molecule than the HB15b mAb.

Other HB15-reactive monoclonal antibodies may be produced using theamino acid sequence disclosed in SEQ ID NO:2, and portions thereoflonger than 8-10 amino acids, using antibody production techniquesdescribed herein and in the literature.

For example, monoclonal antibodies to the protein or a fragment thereofmay be made by the somatic cell hybridization techniques describedinitially by Kohler, B. and Milstein, C., Nature (1975) 256:495-497. Theprocedure involves immunizing a host animal (typically a mouse becauseof the availability of murine myelomas) with the protein.Antibody-producing cells (e.g., peripheral blood lymphocytes,splenocytes) are taken from the immunized host and mixed with a suitabletumor fusion partner in a liquid growth medium containing a fusogen suchas polyethylene glycol of molecular weight 2000 to 5000. After thefusion the cells are washed to remove residual fusion medium andincubated in a selective growth medium (i.e., a growth medium containingadditives to which the parent tumor line is sensitive) such as HATmedium. Surviving hybrids may be expanded and their culture mediascreened for the presence of antibodies by radioimmunoassay (RIA).Positive cultures may be screened for their ability to recognize andbind to the protein by immunoprecipitating labeled cell extracts withthe positive cultures and analyzing the precipitate by SDS-PAGE for thepresence of a labeled component. Hybrids that produce antibody thatbinds specifically to the protein may be subcloned and grown in vitro orin vivo by known procedures. The antibody may be isolated from theresulting culture medium or body fluid, as the case may be, byconventional procedures for isolating immunoglobulins.

Thus, monoclonal antibodies may be made against multiple epitopes of theHB15 polypeptide or an HB15 mammalian homolog.

EXAMPLE V

Detection of HB15 expression.

1. Immunoprecipitation of cell surface HB15

In order to detect the presence of HB15 or an HB15 homolog on certaincell types, an anti-HB15 monoclonal antibody may be used toimmunoprecipitate the cognate antigen from a given cell type, asfollows.

The anti-HB15a mAb was purified, coupled to beads and used toimmunoprecipitate HB15 from detergent solubilized extracts ofsurface-iodinated cell lines, as follows. Cells were washed twice,resuspended in saline and labeled by the iodogen method as described(Thompson et al., Biochem. 24:743-750 (1987)). After washing, the cellswere lysed in 1 ml of buffer containing 1% (v/v) TRITON X-100 andprotease inhibitors as described (Tedder et al., Proc. Natl. Acad. Sci.,USA 85:208 (1988)). Immunoprecipitations were carried out usinganti-HB15a mAb or mouse Ig (as a negative control) directly conjugatedto AFFIGEL (BioRad, Richmond, Va.) at 2 mg of mAb per ml of gelaccording to the manufacturer's instructions. Cell lysates wereprecleared twice for 2 hours using 50 μ (50% v/v) of murine Ig coatedbeads at 4° C. Cell lysates were precleared again overnight. Half of theprecleared lysate was then incubated with 25 μ of anti-HB15a mAb-coatedbeads or murine Ig-coated beads with constant rotation at 4° C. for 18hours. Immunoprecipitates were washed and analyzed by SDS-PAGE asdescribed (Tedder et al., Proc. Natl. Acad. Sci., USA 85:208 (1988))with half of the sample run in the presence of 5% 2-mercaptoethanol(reducing conditions). M_(r) were determined using pre-stained standardmolecular weight markers (Gibco/BRL).

Optimum results were obtained using the K562-HB15 cell line (K562 cellstransfected with pHB15 cDNA) since the level of HB15 expression washigher than in other cell lines. The anti-HB15a mAb specificallyimmunoprecipitated proteins that migrated as a single broad band of˜45,000 M_(r). Similar results were obtained when the immunoprecipitatedmaterials were run under reducing or nonreducing conditions. A similarprotein was immunoprecipitated from the Raji cell line except the M_(r)was ˜40,000. Thus, HB15 was expressed as a noncovalently-associatedsingle chain molecule on the cell surface.

2. HB15 is expressed by activated lymphocytes

In order to determine the tissue distribution of HB15 or an HB15homolog, an anti-HB15 monoclonal antibody may be used to identify thepresence of the cognate antigen by immunofluorescence staining and/orimmunohistological analysis of different tissues, as follows. Cells werekept at 4° C. and were examined immediately after isolation. Indirectimmunofluorescence analysis of viable cells was carried out afterwashing the cells three times. The cells were then incubated for 20 minon ice with each mAb as ascites fluid diluted to the optimalconcentration for immunostaining. Isotype-matched murine antibodies thatwere unreactive with human leukocytes were used as negative controls.After washing, the cells were treated for 20 min at 4° C. withfluorescein isothiocyanate-conjugated goat anti-mouse Ig antibodies(Southern Biotechnology Associates, Birmingham, Ala.). Single colorimmunofluorescence analysis was performed on an Epics Profile flowcytometer (Coulter Electronics, Hialeah, Fla.). Ten thousand cells wereanalyzed for each sample. All tissues were stained applying amodification of the APAAP procedure as described by Cordell et al., J.Histochem. Cytochem. 31.:219-229 (1984). Basically, the slides werefirst incubated with monoclonal antibody followed by an incubation stepwith rabbit anti-mouse (bridging) antibody. Subsequently, a monoclonalantibody against alkaline phosphatase pre-incubated with alkalinephosphatase was applied. In order to enhance the sensitivity of thisprocedure, the number of phosphatase molecules on the surface wasincreased by using one or two layers of bridging antibody andanti-phosphatase antibody. Bound phosphatase molecules were visualizedusing new fuchsin as a substrate (Cordell et al., J. Histochem.Cytochem. 31:219-229 (1984)).

The tissue distribution of the HB15 surface antigen was examined byindirect immunofluorescence staining with flow cytometry analysis. Twocell lines that did not express HB15 message were transfected with thepHB15 cDNA subcloned into the Bam HI site of the retroviral vectorPZIPNEOSV(X). Referring to FIG. 4, the immunofluorescence resultsobtained with three lymphoblastoid cell lines that express HB15 aredemonstrated. The open histograms show the cellular reactivity with theHB15a antibody, and the shaded histograms demonstrate background levelsof immunofluorescence staining obtained with unreactive controlantibodies. Among 33 cell lines examined, HB15 was expressed atdetectable levels by B cell lines (including Raji, Daudi, Namalwa,Arent, BJAB, SB, Jijoy, Akata, and SLA) and T cell lines (includingJurkat, H-9, Rex, H-SB2, and Hut-78). However, HB15 expression wasgenerally low and variable. The highest levels of cell-surfaceexpression were always obtained where the cell cultures were recentlysplit and were thus proliferating maximally. Cell lines that did notexpress detectable levels of HB-15 included: K562; the B cell linesNALM-6 and Ramos; the T cell lines, MOLT-3, RPMI 8405, PEER, MOLT-14,CEM and HPB-ALL; the myelomonocytic line, HL60; the natural killer cellline, YT; the colon carcinoma lines, Colo-205 and HT29; the lung celllines, NCI-H69, and NCI-H82, the prostate line, PC3; the melanoma line,MEWO; and the breast tumor lines, ZRT5.1, MCF7 and BT20.

Expression of HB15 by normal blood leukocytes was also examined. Humanblood was obtained by protocols approved by the Human ProtectionCommittee of Dana-Farber Cancer Institute and mononuclear cells wereisolated by Ficoll-Hypaque density gradient centrifugation. Mononuclearcells (10⁶ /ml) in complete media (RPMI-1640 supplemented with 15% fetalcalf serum, antibiotics and glutamine) were stimulated withphytohemagglutinin-P (2 μg/ml; Difco, Detroit, Mich.), Con A (10 μg/ml,Miles Laboratories, Elkhart, Ind.), pokeweed mitogen (10 μg/ml,Gibco/BRL, Bethesda, Md.) or phorbol myristate 13-acetate (PMA, 10ng/ml, Sigma, St. Louis, Mo.) as described (Tedder et al., J. Immunol.144:532-540 (1990)). Lymphocytes were harvested at the indicated timepoints, washed once in complete media, and aliquoted for immediateimmunofluorescence staining.

Cell-surface expression of HB15 was not detected at significant levelson circulating lymphocytes, natural killer cells or monocytes in 15blood samples. Therefore, the possibility that HB15 was expressedfollowing cellular activation was examined by inducing T lymphocyteproliferation with the mitogens concanavalin A (ConA), pokeweed mitogen,phytohemag-glutinin-P or phorbol esters (PMA). Expression of HB15 wasexamined 2, 8, 12, 24, 48, 72, 120 and 240 hours following theinitiation of cultures. Appearance of HB15 expression paralleledcellular proliferation such that optimal expression was on days 3through 5 following the initiation of cultures. Also, the quantity ofHB15 expression induced was not correlated with any specific mitogen,but correlated more with the strength of the mitogenic signal such thatcell-surface expression was predominantly found on the larger blastcells. Therefore, HB15 was expressed by lymphocytes followingactivation.

3. Immunohistological analysis of HB15 expression

The lymphocyte specificity and tissue distribution of HB15 was alsoexamined by immunohistological analysis of different human tissues.Basically, the anti-HB15a mAb was used to stain thymus, tonsil, spleen,lymph node, kidney, renal pelvis and ureter, Fallopian tube, liver,pancreas, stomach, breast, lung, esophagus, skeletal muscle, skin,uterus, salivary gland, thyroid gland, adrenal gland, heart, appendixand colon. (Referring to FIGS. 5A-5F), in most cases, HB15 expressionappeared lymphocyte specific in that no significant reactivity wasobserved in non-lymphoid tissues. Among tonsil and lymph nodes (FIG.5A), HB15 was expressed reasonably strongly by scattered cells inintrafollicular regions (T cell zones) (FIG. 5C). Although some of thesecells may have been lymphoblasts, most were interdigitating reticulumcells (a subpopulation of dendritic cells) since they appeared largerthan resting lymphocytes and expressed the CD1 surface molecule (FIG.5D). Also, some cells (50-80%) within germinal centers (GC; FIGS. 5A and5B) and follicular mantle zones (FM; FIG. 5A), with the morphology oflymphocytes, were weakly HB15⁺. Among spleen, the HB15⁺ cells werepredominantly restricted to the white pulp, whereas the red pulpremained largely negative. Again, these large, scattered positive cellsin the white pulp are likely to be interdigitating reticulum cells orlymphoblasts. Cortical thymocytes were HB15 negative, while a smallsubpopulation of medullary cells, presumably thymocytes, was positive(FIG. 5E). Unlike other non-hematopoietic tissues, analysis of skinrevealed that some cells with the characteristic scattered branchingmorphology of Langerhans cells (a subpopulation of dendritic cells)expressed HB15 at detectable levels (FIG. 5F). Among allnon-hematopoietic tissues, where inflammatory infiltrations wereapparent, a few scattered lymphocytes were found to express HB15. It isalso likely that circulating dendritic cells are HB15⁺, but because oftheir low frequency they were not readily detected. Similarly, it isalso likely that the malignant counterparts of dendritic cells expressHB15 and that this molecule can be used as a diagnostic marker formalignant cells as the L428 cell line, which is a neoplastic cell linethat was derived from Hodgkin's disease and may representinterdigitating reticulum cells (Schaadt et al., Int. J. Cancer26:723-731 (1980)), is HB15 positive.

It is to be understood that an HB15 homolog, like HB15 itself, willresemble HB15 in its tissue distribution pattern. That is, an HB15homolog will be present on activated lymphocytes and generally absent oninactivated lymphocytes, although the presence or absence of the homologon specific cell lines may not be directly correlated with the presenceor absence of HB15 on such cell lines.

EXAMPLE VI

Quantitation of HB15 Levels.

Endogenous levels of HB15 polypeptide or an HB15 polypeptide homolog inserum can be quantitated using the monoclonal antibodies that have beenproduced against HB15 according to any one of a number of quantitationmethods known to those of ordinary skill in the art, including anenzyme-linked immunoassay (ELISA). For example, a serum sample may beobtained and serially diluted prior to analysis. The dilutions may beassayed in a conventional ELISA wherein the detecting antibody is ananti-HB15 antibody described herein. Detection and quantitation of HB15in the serum sample are performed as described in art.

Uses

The HB15 protein or immunospecific fragments thereof, or antibodies orother antagonists to HB15 function, have a variety of uses, some ofwhich are described below.

1. HB15 as a Marker for Non-follicular Dendritic Cells.

There are at present no specific markers for non-follicular dendriticcells in humans. Use of HB15 monoclonal antibody to identify HB15⁺ cellspermits the isolation and purification of cells expressing this proteinfrom a population of unrelated cells.

2. HB15 as a Marker for Cell Sarcomas and Malignant Phenotypes.

The HB15 monoclonal antibody will also be useful for evaluation anddiagnosis of interdigitating cell sarcomas or other malignant cell typesexpressing this antigen. Therefore, HB15-based agents may be suitablefor immunotherapy or immunoimaging. HB15 protein or immunospecificfragments thereof, or antibodies which antagonize HB15 function areuseful for diagnosis or treatment of a variety of immunologicaldisorders. For such purposes, the soluble external domain may beemployed, typically but not necessarily, polymerized in a multivalentstate using, e.g., dextran or polyamino acid carriers or fusion proteinsof HB15 fragments and carrier molecules. Alternatively, liposomes may beemployed as the therapeutic vehicle, in which case the transmembranedomain and preferably at least some of the cytoplasmic domain will alsobe included.

For example, since Langerhans cells are the primary immunocompetent cellin the skin, playing a role in the presentation of antigen to T cellsand the induction of contact hypersensitivity, and since HB15 isexpressed by Langerhans cells and may be involved in antigenpresentation, it is likely to be involved in the pathogenesis of humanskin disease such as psoriasis, autoimmune disorders, organ transplantand AIDS. Therefore, antagonists to HB15 function can provide importanttherapeutic agents for treatment of these diseases.

Similarly, since HB15 may serve as an accessory molecule for lymphocyteactivation, the HB15 antigen, fragments or domains thereof, may be usedas agonists that would augment or inhibit an immune response.

More specifically, the dendritic cell is a primary target of the humanimmunodeficiency virus, the causative agent of AIDS. It has recentlybeen proposed that 80% of AIDS virus in vivo is produced by dendriticcells, particularly by Langerhans cells, circulating dendritic cells andinterdigitating reticulum cells (Langhoff et al., Proc. Natl. Acad. Sci.USA 88:7998-8002 (1991)). Also, most infections occur through mucosalsurfaces where it is thought that dendritic cells are first infected.Therefore, this reagent provides us with a critical tool for thepotential prevention or treatment of AIDS or AIDS related disorders.

Certain clinical conditions may be monitored using in vitro assays toquantitate the levels of endogenous soluble HB15 in a patient's bloodserum. Based on the finding that several receptors are now known to beshed during various normal and pathological conditions, it is possiblethat HB15 is also lost from the cell surface by an enzymatic process.Also, quantitative detection can be useful in a method of identifyingleukocytes with abnormal or decreased expression of HB15 for diagnosisand/or detection of leukocyte activation or altered leukocyte function.Additionally, the ability to quantitate the amount of receptor, orfragment thereof, produced during the manufacture of a recombinanttherapeutic agent will be advantageous for determining the efficacy ofthe agent.

Similarly, in treating certain clinical conditions, it may be advisableto remove endogenous soluble HB15 or HB15⁺ cells from a patient's blood.This can be done with existing on-line and off-line techniques byemploying immunoselection columns containing antibodies or other bindingagents directed against the disclosed external domain of HB15.

While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

Deposits

The following hybridomas were deposited on Mar. 17, 1992, with theAmerican Type Culture Collection (ATCC) under the terms of the BudapestTreaty.

    ______________________________________                                        Identification        ATCC Designation                                        ______________________________________                                        Anti-HB15a Hybridoma cell line, HB15a                                                               HB 10987                                                Anti-HB15b Hybridoma cell line, HB15b                                                               HB 10988                                                ______________________________________                                    

Applicants' assignee, Dana-Farber Cancer Institute, Inc., representsthat the ATCC is a depository affording permanence of the deposit andready accessibility thereto by the public if a patent is granted. Allrestrictions on the availability to the public of the material sodeposited will be irrevocably removed upon the granting of a patent. Thematerial will be available during the pendency of the patent applicationto one determined by the Commissioner to be entitled thereto under 37CFR 1.14 and 35 USC 122. The deposited material will be maintained withall the care necessary to keep it viable and uncontaminated for a periodof at least five years after the most recent request for the furnishingof a sample of the deposited microorganism, and in any case, for aperiod of at least thirty (30) years after the date of deposit or forthe enforceable life of the patent, whichever period is longer.Applicants' assignee acknowledges its duty to replace the deposit shouldthe depository be unable to furnish a sample when requested due to thecondition of the deposit.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 15                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2272 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 11..625                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCCGCCATGTCGCGCGGCCTCCAGCTTCTGCTCCTGAGCTGCGCC49                           MetSerArgGlyLeuGlnLeuLeuLeuLeuSerCysAla                                       1510                                                                          TACAGCCTGGCTCCCGCGACGCCGGAGGTGAAGGTGGCTTGCTCCGAA97                            TyrSerLeuAlaProAlaThrProGluValLysValAlaCysSerGlu                              152025                                                                        GATGTGGACTTGCCCTGCACCGCCCCCTGGGATCCGCAGGTTCCCTAC145                           AspValAspLeuProCysThrAlaProTrpAspProGlnValProTyr                              30354045                                                                      ACGGTCTCCTGGGTCAAGTTATTGGAGGGTGGTGAAGAGAGGATGGAG193                           ThrValSerTrpValLysLeuLeuGluGlyGlyGluGluArgMetGlu                              505560                                                                        ACACCCCAGGAAGACCACCTCAGGGGACAGCACTATCATCAGAAGGGG241                           ThrProGlnGluAspHisLeuArgGlyGlnHisTyrHisGlnLysGly                              657075                                                                        CAAAATGGTTCTTTCGACGCCCCCAATGAAAGGCCCTATTCCCTGAAG289                           GlnAsnGlySerPheAspAlaProAsnGluArgProTyrSerLeuLys                              808590                                                                        ATCCGAAACACTACCAGCTGCAACTCGGGGACATACAGGTGCACTCTG337                           IleArgAsnThrThrSerCysAsnSerGlyThrTyrArgCysThrLeu                              95100105                                                                      CAGGACCCGGATGGGCAGAGAAACCTAAGTGGCAAGGTGATCTTGAGA385                           GlnAspProAspGlyGlnArgAsnLeuSerGlyLysValIleLeuArg                              110115120125                                                                  GTGACAGGATGCCCTGCACAGCGTAAAGAAGAGACTTTTAAGAAATAC433                           ValThrGlyCysProAlaGlnArgLysGluGluThrPheLysLysTyr                              130135140                                                                     AGAGCGGAGATTGTCCTGCTGCTGGCTCTGGTTATTTTCTACTTAACA481                           ArgAlaGluIleValLeuLeuLeuAlaLeuValIlePheTyrLeuThr                              145150155                                                                     CTCATCATTTTCACTTGTAAGTTTGCACGGCTACAGAGTATCTTCCCA529                           LeuIleIlePheThrCysLysPheAlaArgLeuGlnSerIlePhePro                              160165170                                                                     GATTTTTCTAAAGCTGGCATGGAACGAGCTTTTCTCCCAGTTACCTCC577                           AspPheSerLysAlaGlyMetGluArgAlaPheLeuProValThrSer                              175180185                                                                     CCAAATAAGCATTTAGGGCTAGTGACTCCTCACAAGACAGAACTGGTA625                           ProAsnLysHisLeuGlyLeuValThrProHisLysThrGluLeuVal                              190195200205                                                                  TGAGCAGGATTTCTGCAGGTTCTTCTTCCTGAAGCTGAGGCTCAGGGGTGTGCCTGTCTG685               TTACACTGGAGGAGAGAAGAATGAGCCTACGCTGAAGATGGCATCCTGTTTTGAAGTCCT745               TCACCTCACTGAAAACATCTGGAAGGGGATCCCACCCCATTTTCTGTGGGCAGGCCTCGA805               AAACCATCACATGACCACATAGCATGAGGCCACTGCTGCTTCTCCATGGCCACCTTTTCA865               GCGATGTATGCAGCTATCTGGTCAACCTCCTGGACATTTTTTCAGTCATATAAAAGCTAT925               GGTGAGATGCAGCTGGAAAAGGGTCTTGGGAAATATGAATGCCCCCAGCTGGCCCGTGAC985               AGACTCCTGAGGACAGCTGTCCTCTTCTGCATCTTGGGGACATCTCTTTGAATTTTCTGT1045              GTTTTGCTGTACCAGCCCAGATGTTTTACGTCTGGGAGAAATTGACAGATCAAGCTGTGA1105              GACAGTGGGAAATATTTAGCAAATAATTTCCTGGTGTGAAGGTCCTGCTATTACTAAGGA1165              GTAATCTGTGTACAAAGAAATAACAAGTCGATGAACTATTCCCCAGCAGGGTCTTTTCAT1225              CTGGGAAAGACATCCATAAAGAAGCAATAAAGAAGAGTGCCACATTTATTTTTATATCTA1285              TATGTACTTGTCAAAGAAGGTTTGTGTTTTTCTGCTTTTGAAATCTGTATCTGTAGTGAG1345              ATAGCATTGTGAACTGACAGGCAGCCTGGACATAGAGAGGGAGAAGAAGTCAGAGAGGGT1405              GACAAGATAGAGAGCTATTTAATGGCCGGCTGGAAATGCTGGGCTGACGGTGCAGTCTGG1465              GTGCTCGTCCACTTGTCCCACTATCTGGGTGCATGATCTTGAGCAAGTTCCTTCTGGTGT1525              CTGCTTTCTCCATTGTAAACCACAAGGCTGTTGCATGGGCTAATGAAGATCATATACGTG1585              AAAATTCTTTGAAAACATATAAAGCACTATACAGATTCGAAACTCCATTGAGTCATTATC1645              CTTGCTATGATGATGGTGTTTTGGGGATGAGAGGGTGCTATCCATTTCTCATGTTTTCCA1705              TTGTTTGAAACAAAGAAGGTTACCAAGAAGCCTTTCCTGTAGCCTTCTGTAGGAATTCCT1765              TTTGGGGAAGTGAGGAAGCCAGGTCCACGGTCTGTTCTTGAAGCAGTAGCCTAACACACT1825              CCAAGATATGGACACACGGGAGCCGCTGGGCAGAAGGGACTTCACGAAGGTTTGCATGGA1885              TGTTTTAGCCATTGTTGGCTTTCCCTTATCAAACTTGGGCCCTTCCCTTCTTGGTTTCCA1945              AAGGCATTTTATTGCTTGAGTTATATGTTCACTGTCCCCCTAATATTAGGGAGTAAAACG2005              GATACCAAGTTGATTTAGTGTTTTTACCTCTGTCTTGGCTTTCATGTTATTAAACTGATG2065              CATGTGAAGAAAGGGTGTTTTTCTGTTTTATATTCAACTCATAAGACTTTGGGATAGGAA2125              AAATGAGTAATGGTTACTAGGCTTAATACCTGGGTGATTACATAATCTGTACAATGAACC2185              CCCATGATGTAAGTTTACCTATGTAACAAACCTGCACTTATACCCATGAACTTAAAATGA2245              AAGTTAAAAATAAAAAACATATACAAA2272                                               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 205 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerArgGlyLeuGlnLeuLeuLeuLeuSerCysAlaTyrSerLeu                              151015                                                                        AlaProAlaThrProGluValLysValAlaCysSerGluAspValAsp                              202530                                                                        LeuProCysThrAlaProTrpAspProGlnValProTyrThrValSer                              354045                                                                        TrpValLysLeuLeuGluGlyGlyGluGluArgMetGluThrProGln                              505560                                                                        GluAspHisLeuArgGlyGlnHisTyrHisGlnLysGlyGlnAsnGly                              65707580                                                                      SerPheAspAlaProAsnGluArgProTyrSerLeuLysIleArgAsn                              859095                                                                        ThrThrSerCysAsnSerGlyThrTyrArgCysThrLeuGlnAspPro                              100105110                                                                     AspGlyGlnArgAsnLeuSerGlyLysValIleLeuArgValThrGly                              115120125                                                                     CysProAlaGlnArgLysGluGluThrPheLysLysTyrArgAlaGlu                              130135140                                                                     IleValLeuLeuLeuAlaLeuValIlePheTyrLeuThrLeuIleIle                              145150155160                                                                  PheThrCysLysPheAlaArgLeuGlnSerIlePheProAspPheSer                              165170175                                                                     LysAlaGlyMetGluArgAlaPheLeuProValThrSerProAsnLys                              180185190                                                                     HisLeuGlyLeuValThrProHisLysThrGluLeuVal                                       195200205                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2197 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 45..626                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ACCCACGCGTCCGCCCACGCGTCCGGTGTCGCAGCGCTCCAGCCATGTCGCAAGGC56                    MetSerGlnGly                                                                  CTCCAGCTCCTGTTTCTAGGCTGCGCTGCCTGGCACCGCGATGGCGAT104                           LeuGlnLeuLeuPheLeuGlyCysAlaAlaTrpHisArgAspGlyAsp                              5101520                                                                       GTGGAGGTGACGGTGGCTTGCTCCGAGACTGCCGACTTGCCTTGCACA152                           ValGluValThrValAlaCysSerGluThrAlaAspLeuProCysThr                              253035                                                                        GCGCCCTGGGACCCGCAGCTCTCCTATGCAGTGTCCTGGGCCAAGGTC200                           AlaProTrpAspProGlnLeuSerTyrAlaValSerTrpAlaLysVal                              404550                                                                        TCCGAGAGTGGCACTGAGAGTGTGGAGCTCCCGGAGAGCAAGCAAAAC248                           SerGluSerGlyThrGluSerValGluLeuProGluSerLysGlnAsn                              556065                                                                        AGCTCCTTCGAGGCCCCCAGGAGAAGGGCCTATTCCCTGACGATCCAA296                           SerSerPheGluAlaProArgArgArgAlaTyrSerLeuThrIleGln                              707580                                                                        AACACTACCATCTGCAGCTCGGGCACCTACAGGTGTGCCCTGCAGGAG344                           AsnThrThrIleCysSerSerGlyThrTyrArgCysAlaLeuGlnGlu                              859095100                                                                     CTCGGAGGGCAGCGCAACTTGAGCGGCACCGTGGTTCTGAAGGTGACA392                           LeuGlyGlyGlnArgAsnLeuSerGlyThrValValLeuLysValThr                              105110115                                                                     GGATGCCCCAAGGAAGCTACAGAGTCAACTTTCAGGAAGTACAGGGCA440                           GlyCysProLysGluAlaThrGluSerThrPheArgLysTyrArgAla                              120125130                                                                     GAAGCTGTGTTGCTCTTCTCTCTGGTTGTTTTCTACCTGACACTCATC488                           GluAlaValLeuLeuPheSerLeuValValPheTyrLeuThrLeuIle                              135140145                                                                     ATTTTCACCTGCAAATTTGCACGACTACAAAGCATTTTCCCAGATATT536                           IlePheThrCysLysPheAlaArgLeuGlnSerIlePheProAspIle                              150155160                                                                     TCTAAACCTGGTACGGAACAAGCTTTTCTTCCAGTCACCTCCCCAAGC584                           SerLysProGlyThrGluGlnAlaPheLeuProValThrSerProSer                              165170175180                                                                  AAACATTTGGGGCCAGTGACCCTTCCTAAGACAGAAACGGTA626                                 LysHisLeuGlyProValThrLeuProLysThrGluThrVal                                    185190                                                                        TGAGTAGGATCTCCACTGGTTTTTACAAAGCCAAGGGCACATCAGATCAGTGTGCCTGAA686               TGCCACCCGGACAAGAGAAGAATGAGCTCCATCCTCAGATGGCAACCTTTCGAAGTCCTT746               CACCTGACAGTGGGCTCCACACTACTCCCTGACACAGGGTCTTGAGCACCATCATATGAT806               CACGAAGCATGGAGTATCACCGCTTCTCTGTGCTGTCAGCTTAATGTTTCATGTGGCTAT866               CTGGTCAACCTCGTGAGTGCTTTTCAGTCATCTACAAGCTATGGTGAGATGCAGGTGAAG926               CAGGGTCATGGGAAATTTGAACACTCTGAGCTGGCCCTGTGACAGACTCCTGAGGACAGC986               TGTCTCTCCTACATCTGGGATACATCTCTTTGAATTTGTCCTGTTTCGTTGCACCAGCCC1046              AGATGTCTCACATCTGGCGGAAATTGACAGGCCAAGCTGTGAGCCAGTGGGAAATATTTA1106              GCAAATAATTTCCAGTGGCGAAGGTCCTGCTATTAGTAAGGAGTATTATGTGTACATAGA1166              AATGAGAGGTCAGTGAACTATTCCCCAGCAGGGCCTTTTCATCTGGAAAAGACATCCACA1226              AAAGCAGCAATACAGAGGGATGCCAGCATTTATTTTTTTAATCTTCATGTATTGTCAAAG1286              AAGAATTTTTCATGTTTTTTCAAAGAAGTGTGTTTCTTTCCTTTTTTAAAATATGAAGGT1346              CTAGTTACATAGCATTGCTACGTACAAGCAGCCTGAGAGAAGATGGAGAATGTTCCTCAA1406              AATAGGGACAGCAAGCTAGAACGACTGTACAGTGCCTGCTGGGAAGGGCAGACAATGGAC1466              TGAGAAACCAGAAGTCTGGCCACAAGATTGTCTGTATGATTCTGGACGAGTCACTTGTGG1526              TTTTCACTCTCTGGTTAGTAAACCAGATAGTTTAGTCTGGGTTGAATACAATGGATGTGA1586              AGTTGCTTGGGGAAAGCTGAATGTAGTGAATACATTGGCAACTCTACTGGGCTGTTACCT1646              GTTGATATCCTAGAGTTCTGGAGCTGAGACGATCGCTGTCATATCTCAGCTTGCCCATCA1706              ATCCAAACACAGGAGGCTACAAAAAGGACATGAGCATGGTCTTCTGTGTGAACTCCTCCT1766              GAGAAACGTGGAGACTGGCTCAGCGCTTTGTGCTCGAAGGACTAATCACAAGTTCTTCGA1826              AGATATGGACCTAGGGGAGCTATTGCGCCACGACAGGAGGAAGTTCTCAGATGTTGCATT1886              GATGTAACATTGTTGCATTTCTTTAATGAGCTGGGCTCCTTCCTCATTTGCTTCCCAAAG1946              AGATTTTGTCCCACTAATGGTGTGCCCATCACCCACACTATGAAAAGTAAAAGGGATGCT2006              GAGCAGATACAGGCTAGTCTTACCTCTCAAGTCCATGACTTTCATGCTATTAAAGAATGC2066              ATGTGAAGAGGTGTGTTCTTCTTTTCTATCTTTAAAATGATCGACTTTAGAGTGAGTGTT2126              TGGGTGCTGAGTGGAGAGTAAGAATGCAGAAATGGTAGTGGTAAATGACTGGCTGCTTCC2186              CGAGGGGATCC2197                                                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetSerGlnGlyLeuGlnLeuLeuPheLeuGlyCysAlaAlaTrpHis                              151015                                                                        ArgAspGlyAspValGluValThrValAlaCysSerGluThrAlaAsp                              202530                                                                        LeuProCysThrAlaProTrpAspProGlnLeuSerTyrAlaValSer                              354045                                                                        TrpAlaLysValSerGluSerGlyThrGluSerValGluLeuProGlu                              505560                                                                        SerLysGlnAsnSerSerPheGluAlaProArgArgArgAlaTyrSer                              65707580                                                                      LeuThrIleGlnAsnThrThrIleCysSerSerGlyThrTyrArgCys                              859095                                                                        AlaLeuGlnGluLeuGlyGlyGlnArgAsnLeuSerGlyThrValVal                              100105110                                                                     LeuLysValThrGlyCysProLysGluAlaThrGluSerThrPheArg                              115120125                                                                     LysTyrArgAlaGluAlaValLeuLeuPheSerLeuValValPheTyr                              130135140                                                                     LeuThrLeuIleIlePheThrCysLysPheAlaArgLeuGlnSerIle                              145150155160                                                                  PheProAspIleSerLysProGlyThrGluGlnAlaPheLeuProVal                              165170175                                                                     ThrSerProSerLysHisLeuGlyProValThrLeuProLysThrGlu                              180185190                                                                     ThrVal                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCCATGTCGCAAGGCCTCCAGCTCC25                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ACACGGTCTCCTGGGTCAAG20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ACCTAAGTGGCAAGGTGATC20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GACAGCACTATCATCAGAAG20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTGCAGCTCGGGCACCTACAGGTG24                                                    (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CTGCAGCTCGGGCACCTACAGGTG24                                                    (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TGCACAGCGTAAAGA15                                                             (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ACTTTTAAGAAATACAGAGCGGAGATTGTCCT32                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GAAATACAGAGCGGAGATTGTCCT24                                                    (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACACTCATCATTTTCACTTGT21                                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AGCTTTTCTTCCAGTCACCTCCCCAA26                                                  __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid comprising at least 20nucleotides that hybridizes under stringent conditions with a region ofthe coding portion of the nucleotide sequence of SEQ ID NO: 1, saidisolated nucleic acid comprising less than a full coding sequence forthe HB15 protein having the amino acid sequence of SEQ ID NO:2.
 2. Theisolated nucleic acid of claim 1, comprising at least 50 nucleotides. 3.The isolated nucleic acid of claim 1, comprising at least 100nucleotides.